Hydrogen Abstraction of Camphor Catalyzed by Cytochrome P450_cam: A QM/MM Study

A combined quantum mechanics and molecular mechanics (QM/MM, QM = UB3LYP-D3, MM = AMBER) method is used to study the hydrogen abstraction reaction in P450_cam catalyzed hydroxylation of camphor in the quartet state. Compared to QM/MM calculations in the literature, this study uses larger basis sets for the most important atoms at the active site and QM/MM Hessian harmonic frequency calculations to determine the standard Gibbs free energy of activation and kinetic isotope effect. The QM/MM covalent boundary is treated with a capping hydrogen atom method, which is simple and robust. An energy barrier of 21.3 kcal/mol and a standard free energy of activation of 16.8 kcal/mol are obtained for this hydrogen abstraction reaction. These values are similar to those reported in the literature, suggesting that when a general protocol is followed, QM/MM results are reproducible. It is found that using a sufficiently large basis set is important to minimize basis set errors

Flexibility of Binding Site is Essential to the Ca²⁺ Selectivity in EF-Hand Calcium-Binding Proteins

High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site

Flexibility of Binding Site is Essential to the Ca²⁺ Selectivity in EF-Hand Calcium-Binding Proteins

High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site

The 8-Silyloxyquinoline Scaffold as a Versatile Platform for the Sensitive Detection of Aqueous Fluoride

Utilizing a novel 8-silyloxyquinoline scaffold, we demonstrate the ability to synthesize fluorogenic probes for the sensitive and selective detection of inorganic fluoride (NaF) in aqueous samples. Our initial probe design (<b>2</b>) is capable of detecting inorganic fluoride at levels as low as 3.8 μM (72 ppb) in aqueous solutions, well below PHS recommended levels for drinking water (0.7–1.2 ppm), placing this probe among the most sensitive fluoride sensors reported to date. Furthermore, our results highlight the utility of the readily modifiable 8-silyloxyquinoline scaffold for the design of tailored fluoride sensing platforms. We demonstrate the ability to rationally tune the fluorescence and physical properties of the 8-silyloxyquinoline scaffold, producing a red-shifted fluoride probe (<b>4</b>) capable of detecting 50 μM (0.95 ppm) NaF in aqueous samples using a straightforward test-strip-based assay format. Taken together this work provides a template for the design of fluoride sensors capable of reporting on relevant concentrations of fluoride in the laboratory and in the field

Improved Photoinduced Fluorogenic Alkene–Tetrazole Reaction for Protein Labeling

The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene–tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene–tetrazole reaction product. This finding provides useful guidance to the future design of alkene–tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene–tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene–tetrazole chemistry in real-time imaging in live cells